Chain length specificity for activation of cPLA2alpha by C1P: use of the dodecane delivery system to determine lipid-specific effects

Abstract

Previously, our laboratory demonstrated that ceramide-1-phosphate (C1P) specifically activated group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) in vitro. In this study, we investigated the chain length specificity of this interaction. C1P with an acyl-chain of >or=6 carbons efficiently activated cPLA(2)alpha in vitro, whereas C(2)-C1P, was unable to do so. Delivery of C1P to cells via the newly characterized ethanol/dodecane system demonstrated a lipid-specific activation of cPLA(2)alpha, AA release, and PGE(2) synthesis (EC(50) = 400 nM) when compared to structurally similar lipids. C1P delivered as vesicles in water also induced a lipid-specific increase in AA release. Mass spectrometric analysis demonstrated that C1P delivered via ethanol/dodecane induced a 3-fold increase in endogenous C1P with little metabolism to ceramide. C1P was also more efficiently delivered (>3-fold) to internal membranes by ethanol/dodecane as compared to vesiculated C1P. Using this now established delivery method for lipids, C(2)-C1P was shown to be ineffective in the induction of AA release as compared with C(6)-C1P, C(16)-C1P, and C(18:1) C1P. Here, we demonstrate that C1P requires >or=6 carbon acyl-chain to activate cPLA(2)alpha. Thus, published reports on the biological activity of C(2)-C1P are not via eicosanoid synthesis. Furthermore, this study demonstrates that the alcohol/dodecane system can be used to efficiently deliver exogenous phospholipids to cells for the examination of specific biological effects.

Fig. 1.

Activation of cPLA₂α by C1P is dependent on the acyl chain length. cPLA₂α activity was measured as a function of PC molar concentration in the absence and presence of 4 mol % D-erythro C₂, C₆, C₁₆, and C_18:1 C1P for 45 min at 37 C as described in Materials and Methods. The PC mole fraction was held constant at 0.137. Data are presented as cPLA₂α activity measured as nanomoles of arachidonic acid produced/minute/milligram of recombinant cPLA₂α ± SE.

Fig. 2.

The effects of natural C1P on AA release and PGE₂ synthesis. A: The effects of ceramide-1-phosphate on AA release is lipid-specific at low doses. A549 cells (5 × 10⁴) were labeled overnight with 5 μCi/ml [³H]AA (5 nM). Cells were washed and placed in DMEM supplemented with 2% fetal bovine serum for 2 h, followed by treatment with 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, and 1 μM of D-e-C_18:1 C1P (▪), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA) (♦), or D-e-C_18:1 ceramide (▴) solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) for 2 h. For quantification of AA release, media was transferred to 1.5 ml polypropylene tubes, centrifuged 10,000 g, and ³H AA determined by scintillation counting. The results are presented as DPM of ³H-AA per ml of media controlled for equivalent number of cells by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Data are representative of 15 separate determinations on 5 separate occasions. B: Natural C1P, but not the structurally similar PA nor ceramide, is capable of inducing PGE₂ synthesis. A549 cells (5 × 10⁴) were washed and placed in DMEM supplemented with 2% FBS for 2 h. Cells were then treated with 0.1, 0.2, 0.4, 0.5, 0.75, and 1 μM D-e-C_18:1 C1P (▪), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA) (♦), or D-e-C_18:1 ceramide (▴) solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) for 2 h. For measurement of PGE₂ levels, media were assayed according to manufacturer s instructions using the Prostaglandin E₂ monoclonal EIA Kit from Cayman Chemical (Ann Arbor, MI, Catalog No. 514010). Briefly, media containing PGE₂ competes with PGE₂ acetylcholinestaerase conjugate for a limited amount of PGE₂ monoclonal antibody. The antibody-PGE₂ conjugate binds to a goat-anti-mouse antibody previously attached to the wells. The plate is washed to remove any unbound reagents and then the substrate to acetylcholinesterase is provided. The concentration of PGE₂ in a sample is inversely proportional to the yellow color produced. The results are presented as picograms of PGE₂ per ml of media controlled for equivalent number of cells by MTT assay. Data are representative of six separate determinations on two separate occasions. C: cPLA₂α translocates specifically in response to C1P. A549 cells (1 × 10⁵) were infected at 10 MOI with an adenoviral construct containing cPLA₂α-GFP. 48 h postinfection, cells were treated with 500 nM D-e-C_18:1 Ceramide (A), 500 nM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (PA) (B), and 500 nM D-e-C_18:1 C1P (C), all solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) for 2 h. cPLA₂α localization was visualized using an Olympus BX50WI confocal microscope at 488 nM (Fluoview detector) using a 40× liquid immersable lens with a 1.5×-enhanced magnification. Data are representative of three separate determinations on two separate occasions.

Fig. 3.

Activation of cPLA₂α by C1P is independent of its delivery medium. [³H]AA (0.20 μCi/ml) labeled NR8383 cells (1 × 10⁶ cells/ml) were treated with C1P (♦), PA (▪), and Ctrl (H₂O) (▴) sonicated in water at 5, 15, and 30 μM for 6 h. For quantification, the media was collected and centrifuged at 10000 g for 5 min and the amount of [³H] labeled AA in the media was quantified by scintillation counting. The results are presented as DPM (dissociations per minute) of radioactivity per ml of media.

Fig. 4.

C1P is efficiently delivered to cells via EtOH/dodecane and is slowly metabolized to ceramide. A549 cells (1 × 10⁶) were treated with 500nM D-e-C_18:1 C1P for 2 h. The cells were then harvested and lipids extracted followed by analysis by mass spectroscopy. Data are presented as pmols of lipid. Inset: Exogenously added C1P is slowly metabolized to ceramide. A549 cells (1 × 10⁶) were treated with 500nM D-e-C_18:1 C1P for 2 h. The cells were then harvested and lipids extracted followed by analysis by mass spectroscopy for C1P and ceramide (inset). Data is presented as pmols lipids. Error bars indicate SD.

Fig. 5.

C1P is efficiently taken up by cells into internal membranes when delivered via EtOH/dodecane. A: A549 cells (1 × 10⁶) were treated for 2 h with radiolabeled 500nM D-e-C_18:1 C1P solubilized in either EtOH/dodecane or by sonication. The cells were then harvested and lysed by freeze thawing. The plasma membranes were separated from the internal membranes and the amounts of radiolabeled lipids in the different fractions measured by scintillation counting. The results are presented as the fold increase of the levels of C1P over background in each fraction for the different methods of delivery. B: C1P delivered via EtOH/dodecane system reaches specific internal membranes with higher efficiency. A549 cells (1 × 10⁶) were treated for 2 h with radiolabeled, 500nM D-e-C_18:1 C1P solubilized in either EtOH/dodecane or by sonication. The cells were then harvested and lysed by freeze thawing and homogenized. The resultant mixture was subjected to subcellular fractionation via differential centrifugation as previously described (26). All fractions were confirmed as previously described by our laboratory (26). The results are presented as a comparison of the levels of C1P in each fraction for the different methods of delivery of C1P. C: Differential centrifugation allows the separation of the different organelles into different subcellular fractions. A₅₄₉ cells were treated and subjected to subcellular fractionation as in 5B to obtain nuclear (N), mitochondrial and trans-Golgi (M,TGN), endoplasmic reticulum (ER), plasma membrane (PM) and cytosolic (Cyt) fractions. All fractions were probed with organelle specific markers to assay for purity of the fractions. Anti-lamin AC (Santa Cruz 1:1000) for nuclear, anti-mitochondrial (AbCam 1:1000), anti-TGN46 for trans-Golgi (AbCam 1:1000), anti-protein disulfide isomerase (PDI) for ER (AbCam 1:1000), and anti-caveolin 1 for plasma membrane (Santa Cruz 1:1000) were used as organelle markers. Note: only the ER fraction demonstrated the proper PDI signal. The chemiluminescence signals observed in the other fractions are nonspecific, and are not present when immunoblotting using purified PDI.

Fig. 6.

Naturally occurring C1P are the best activators of cPLA₂α in vivo. A: A549 cells (5 × 10⁴) were labeled overnight with 5 μCi/ml [³H]AA (5 nM). Cells were washed and placed in DMEM supplemented with 2% FBS for 2 h. Cells were then treated with either EtOH/dodecane alone, media (No C1P) or 500 nM D-erythro C₂, C₆, C₁₆, and C_18:1 C1P solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) as previously described for C1P(7) for 2 h. For quantification of AA release, media was transferred to 1.5 ml polypropylene tubes, centrifuged at 10,000 g, and ³H cpm (counts per minute) determined by scintillation counting. Results were controlled for equivalent number of cells quantified by WST assay. The results are presented as fold increase in AA release when compared with EtOH/dodecane treatment. The results are an average of three experiments ± SD. A, inset: EtOH/dodacane system successfully delivers both the long and short chain C1Ps to cells. A549 cells (1 × 10⁶) were treated for 2 h with 1μM D-e-C₂ C1P or D-e-C_18:1C1P. The cells were then harvested and analyzed by mass spectrometry as previously described. Data are expressed as increase in pmol quantity of the lipid over that of the controls. B: cPLA₂α translocates to the membrane in response to natural long chain C1P but not the short C₂-C1P. A549 cells (1 × 10⁵) were infected at 10 MOI with an adenoviral construct containing cPLA₂α-GFP. 48 h postinfection, cells were treated with 500 nM D-e-C_18:1 C1P or C₂-C1P solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) for 2 h. The cells were subsequently lysed and centrifuged at 100,000 g to separate membranes from the cytosol. Equal total protein from each fraction was subjected to western analysis and probed for the indicated proteins. Data are representative of six separate determinations on two separate occasions. C: Translocation of cPLA₂α in response to C1P is due to a direct interaction with C1P. A549 cells (1 × 10⁵) were infected at 10 MOI with an adenoviral constructs containing wild-type and mutant (R57A/K58A/R59A) cPLA2α-GFP. 48 h postinfection, cells were treated with 1μM D-e-C_18:1 C1P solubilized in 2% dodecane/98% EtOH (final concentration in treatments was 0.002% dodecane/0.098% EtOH) for 3 h. cPLA₂α localization was visualized using an Olympus BX50WI confocal microscope at 488 nM (Fluoview detector) using a 40× liquid immersable lens with a 1.5×-enhanced magnification. Data are representative of three separate determinations on two separate occasions.